1. Field of Invention
The present invention generally relates to electro-cell manipulation. More particularly, the invention concerns an electroporation apparatus and method for generating and applying an electric field according to a user-selected pulsing scheme to more efficiently introduce molecules into cells and minimize damage to cellular tissue.
2. Description of Related Art
A cell has a natural resistance to the passage of molecules through its membranes into the cell cytoplasm. Scientists in the 1970s first discovered "electroporation", where electrical fields are used to create pores in cells without causing permanent damage to them. Electroporation was further developed to aid in the insertion of various molecules into cell cytoplasm by temporarily creating pores in the cells through which the molecules pass into the cell.
Electroporation has been used to implant materials into many different types of cells. Such cells, for example, include eggs, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, and sperm. Furthermore, electroporation has bene used to implant a variety of different materials, referred to herein as "implant materials", "implant molecules", "implant agents". Namely, these materials have included DNA, genes, and various chemical agents.
Electroporation has been used in both in vitro and in vivo procedures to introduce foreign material into living cells. With in vitro applications, a sample of live cells is first mixed with the implant agent and placed between electrodes such as parallel plates. Then, the electrodes apply an electrical field to the cell/implant mixture. Examples of systems that perform in vitro electroporation include the Electro Cell Manipulator ECM 600 product, and the Electro Square Porator T820, both made by the BTX Division of Genetronics, Inc.
With in vivo applications of electroporation, electrodes are provided in a caliper that grips the epidermis overlying a region of cells to be treated. Alternatively, needle-shaped electrodes may be inserted into the patient, to access more deeply located cells. In either case, after the implant agent is injected into the treatment region, the electrodes apply an electrical field to the region. Examples of systems that perform in vivo electroporation include the Electro Cell Manipulator ECM 600 product, and the Electro Square Porator T820, both made by the BTX Division of Genetronics, Inc.
One type of in vivo electroporation application under research is electrochemotherapy, which uses electroporation to deliver chemotherapeutic agents directly into tumor cells. This treatment is carried out by infusing an anticancer drug directly into the tumor and applying an electric field to the tumor between a pair of electrodes. The molecules of the drug are suspended in the interstitial fluid between and in and around the tumor cells. By electroporating the tumor cells, molecules of the drug adjacent many of the cells are forced or drawn into the cell, subsequently killing the cancerous tumor cell.
Electroporation in this application is especially beneficial because electroporation can help minimize the amount of implant agent used, these chemicals frequently being harmful to normal cells. In particular, less of the implant agent can be introduced into the tumorous area because the electroporation will enable more of the implant agent to actually enter the cell. Electroporation is also beneficial for chemotherapy because some of the most promising anti-cancer drugs, such as Bleomycin, normally cannot penetrate the membranes of certain cancer cells. However, recent experiments with electroporation demonstrated that it is possible to insert the Bleomycin directly into the cells.
Known electroporation techniques (both in vitro and in vivo) function by applying a brief high voltage pulse to electrodes positioned around the treatment region. The electric field generated between the electrodes causes the cell membranes to temporarily become porous, whereupon molecules of the implant agent enter the cells. In known electroporation applications, this electric field comprises a single square wave pulse on the order of 1000 V/cm, of about 100 .mu.s duration. Such a pulse may be generated, for example, in known applications of the Electro Square Porator T820, made by the BTX Division of Genetronics, Inc.
Although known methods of electroporation may be suitable for certain applications, the electric field may actually damage the electroporated cells in some cases. For example, an excessive electric field may damage the cells by creating permanent pores in the cell walls. In extreme cases, the electric field may completely destroy the cell.
Attempting to ameliorate these undesirable effects, at least one application has proposed the use of multiple pulses. One application, for example, proposed use of an electromechanical relay to provide consecutive first and second pulses. S. I. Sukharev et al., Biophys. J. Vol. 63, November 1992, pp. 1320-1327. More particularly, Sukharev uses an electric field pulse 100 as shown in FIG. 1. The pulse 100 includes (1) a first, narrow duration, high voltage pulse 102, (2) a delay 103 of .DELTA.t, during which no pulse is generated, then (3) a second, wide duration, low voltage pulse 104. The first pulse 102 was intended to porate the membrane, whereas the second pulse 104 was intended to electrophorese DNA into the cell cytosol. Sukharev recognized that the delay 103 should not be excessive.
Although the Sukharev system may provide satisfactory results in some applications, this system may not be completely adequate for certain other applications. Some users may find, for example, that Sukharev's electroporation does not effectively move enough molecules of the implant agent into the target cells. This results from an excessive delay 103 between Sukharev's first 102 and second 104 pulses, as recognized by the present inventor. The pores of a cell, created by electroporation, stay open for a finite time, largely depending upon the cell's temperature. Thus, the effect of the first pulse may start to significantly decay (thereby closing the cell's pores) during the delay between the first and second pulses. In some applications, this may be sufficient to completely nullify the first pulse's effect upon the cell by the time the second pulse occurs. As a result, the efficacy of Sukharev's electroporation may be insufficient in some cases. Moreover, lacking an effective first pulse, the second pulse of Sukharev's system may need to be increased to the point where it permanently destroys cells.
The delay described above is inherent to the Sukharev system due to the use of electromechanical relays. Sukharev uses independent pulse generators, whose outputs are selectively coupled to output electrodes by a relay. As known in the art, however, the switching of an electromechanical relay typically takes a significant amount of time, sometimes even 50-100 ms. Therefore, the efficacy of the implant agent achieved by Sukharev may be too low for some applications.
Thus, as recognized by the present inventor, existing electroporation systems may not be suitable for certain applications due to the generation of an excessive electric field, or due to the delay between adjacent pulses. Furthermore, many existing electroporation systems lack sufficient control over the parameters of the electric field pulses such as amplitude, duration, number of pulses, etc.